發(fā)布時間：2018-09-04 10:53

【摘要】：免疫系統(tǒng)是機(jī)體在進(jìn)化過程中形成的一種可以通過多種屏障來識別“自己”和“非己”的系統(tǒng)。黏膜免疫是抵抗外源入侵的第一道屏障。當(dāng)“非己”成分入侵機(jī)體后可以被機(jī)體的模式識別受體(PRRs)所識別,并引起信號轉(zhuǎn)導(dǎo),從而促進(jìn)某些核轉(zhuǎn)錄因子的激活并入核進(jìn)而調(diào)節(jié)相關(guān)基因的表達(dá)。其中第一個被確認(rèn)的模式識別受體就是TLRs家族。本研究一方面通過構(gòu)建TGEV結(jié)構(gòu)蛋白和非結(jié)構(gòu)蛋白真核表達(dá)載體研究其對細(xì)胞FcRn表達(dá)的影響,并挖掘病毒相關(guān)蛋白的功能域;另一方面研究TGEV、UV-TGEV以及TGEV蛋白引起細(xì)胞FcRn表達(dá)上調(diào)的相關(guān)TLRs信號通路。取得的主要研究成果如下:1.TGEV對細(xì)胞FcRn表達(dá)的影響TGEV感染PK-15細(xì)胞或IPEC-J2細(xì)胞后,通過熒光定量PCR、雙熒光素酶報告系統(tǒng)以及Western Blot實驗對細(xì)胞FcRn的mRNA和蛋白表達(dá)進(jìn)行檢測。結(jié)果表明TGEV能引起細(xì)胞FcRn mRNA和蛋白表達(dá)顯著上調(diào)。2.TGEV蛋白對細(xì)胞FcRn表達(dá)的影響本研究通過紫外線滅活TGEV,并用不同劑量的UV-TGEV刺激PK-15細(xì)胞后通過雙熒光素酶報告系統(tǒng)和Western Blot檢測發(fā)現(xiàn)UV-TGEV能夠顯著上調(diào)FcRn mRNA和蛋白表達(dá)水平。說明TGEV的結(jié)構(gòu)蛋白參與了細(xì)胞FcRn表達(dá)的調(diào)控。與TGEV活病毒刺激相比,UV-TGEV上調(diào)細(xì)胞FcRn程度較低,這說明除病毒核酸以外,TGEV的非結(jié)構(gòu)蛋白也可能參與了對FcRn的表達(dá)調(diào)控。3.TGEV蛋白真核表達(dá)載體的構(gòu)建本研究通過提取TGEV WH-1株的RNA,反轉(zhuǎn)錄獲得cDNA,并以此為模板,應(yīng)用TGEV各蛋白基因的特異性引物分別進(jìn)行擴(kuò)增,并將擴(kuò)增產(chǎn)物回收后經(jīng)酶切連接到pCAGGS-HA真核表達(dá)載體,通過PCR擴(kuò)增、酶切鑒定及測序驗證準(zhǔn)確后,再分別將各蛋白重組表達(dá)質(zhì)粒轉(zhuǎn)染PK-15細(xì)胞。通過熒光定量PCR和Western Blot檢測FcRn的表達(dá)水平。結(jié)果表明結(jié)構(gòu)蛋白M、N和E以及非結(jié)構(gòu)蛋白nsp1、nsp5、nsp7、nsp8、nsp9、nsp15均可以上調(diào)細(xì)胞FcRn的表達(dá)。4.TGEV M和N蛋白截短表達(dá)載體的構(gòu)建為了探究M蛋白和N蛋白調(diào)控FcRn表達(dá)的結(jié)構(gòu)功能域,本研究通過構(gòu)建不同的截短蛋白表達(dá)質(zhì)粒并轉(zhuǎn)染PK-15細(xì)胞,通過Western Blot和熒光定量PCR實驗對細(xì)胞FcRn表達(dá)水平進(jìn)行檢測。最終確定N蛋白有效功能域位于156-198位氨基酸,M蛋白有效功能域位于219-262位氨基酸。5.探究TGEV上調(diào)FcRn表達(dá)的相關(guān)TLRs信號通路本研究對TGEV、UV-TGEV以及TGEV蛋白上調(diào)細(xì)胞FcRn的表達(dá)是否通過激活TLRs相關(guān)通路進(jìn)行調(diào)控展開了研究。首先將MyD88和TRIF負(fù)調(diào)控質(zhì)粒轉(zhuǎn)染PK-15細(xì)胞,再應(yīng)用TGEV和UV-TGEV刺激細(xì)胞,然后通過檢測細(xì)胞FcRn表達(dá)水平的變化,證實了TGEV和UV-TGEV均能通過TLRs通路上調(diào)細(xì)胞FcRn蛋白的表達(dá);通過熒光定量PCR檢測證實了TGEV感染細(xì)胞能夠激活TLR1、TLR2、TLR3、TLR4和TLR6的表達(dá),UV-TGEV能夠激活TLR1和TLR2。進(jìn)一步將能上調(diào)FcRn表達(dá)的各種TGEV蛋白表達(dá)質(zhì)粒分別轉(zhuǎn)染細(xì)胞后發(fā)現(xiàn)nsp1、nsp5、nsp7、nsp8、nsp9、nsp15等六種非結(jié)構(gòu)蛋白后均能顯著激活TLR1、TLR2、TLR4和TLR6,結(jié)構(gòu)蛋白E能顯著激活TLR2、TLR4和TLR6,截短蛋白N1能顯著激活TLR1、TLR4和TLR6,截短蛋白M2能激活TLR2和TLR4。
[Abstract]:Mucosal immunity is the first barrier against foreign invasion. When "non-self" components invade the body, they can be recognized by the body's pattern recognition receptors (PRRs) and cause signal transduction, thereby promoting the body's ability to recognize itself and its non-self. Some nuclear transcription factors are activated and incorporated into the nucleus to regulate the expression of related genes. The first recognized pattern recognition receptor is the TLRs family. On the one hand, the up-regulation of FcRn expression induced by TGEV, UV-TGEV and TGEV proteins was studied. The main results were as follows: 1. The effect of TGEV on FcRn expression in PK-15 cells or IPEC-J2 cells infected with TGEV was studied by fluorescence quantitative PCR, double luciferase reporting system and Western Blot assay. The results showed that TGEV could induce a significant up-regulation of FcRn mRNA and protein expression. 2. The effect of TGEV protein on FcRn expression in PK-15 cells was studied by UV inactivation of TGEV and stimulation of PK-15 cells with different doses of UV-TGEV. The expression level of FcRn mRNA and protein was up-regulated, indicating that the structural protein of TGEV was involved in the regulation of FcRn expression. Compared with the stimulation of TGEV, the level of FcRn up-regulated by UV-TGEV was lower, indicating that the non-structural protein of TGEV might also be involved in the regulation of FcRn expression besides viral nucleic acid. In this study, the RNA of TGEV WH-1 strain was extracted, and the cDNA was obtained by reverse transcription. As a template, the specific primers of TGEV protein genes were used to amplify the amplified products respectively. The recombinant products were then digested and linked to pCAGGS-HA eukaryotic expression vector by enzyme digestion, PCR amplification, enzyme digestion and sequencing to verify their accuracy. The expression of FcRn in PK-15 cells was detected by fluorescence quantitative PCR and Western Blot. The results showed that structural proteins M, N and E, as well as non-structural proteins nsp1, nsp5, nsp7, nsp8, Nsp9 and nsp15 could up-regulate the expression of FcRn in PK-15 cells. In this study, different truncated protein expression plasmids were constructed and transfected into PK-15 cells. The expression level of FcRn was detected by Western Blot and fluorescence quantitative PCR. The effective domain of N protein was located at 156-198 amino acids and the effective domain of M protein was located at 219-262 amino acids. 5. To explore the up-regulation of FcRn by TGEV. In this study, we investigated whether the up-regulation of FcRn expression by TGEV, UV-TGEV and TGEV proteins was mediated by activation of TLRs-related pathways. It was confirmed that both TGEV and UV-TGEV could up-regulate the expression of FcRn protein through the TLRs pathway, and that TGEV-infected cells could activate the expression of TLR1, TLR2, TLR3, TLR4 and TLR6, and that UV-TGEV could activate TLR1 and TLR2, and further up-regulate the expression of various TGEV protein plasmids in FcRn-infected cells. TLR1, TLR2, TLR4 and TLR6 were significantly activated by p1, nsp5, nsp7, nsp8, Nsp9 and nsp15. Structural protein E could significantly activate TLR2, TLR4 and TLR6, truncated protein N1 could significantly activate TLR1, TLR4 and TLR6, truncated protein M2 could activate TLR2 and TLR4.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

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